DESIGN AND SYNTHESIS OF NOVEL SYNTHETIC ANTIOXIDANTS FOR THE TREATMENT OF OXIDATIVE STRESS RELATED DISEASES

Free radicals play an important role in many physiological processes that occur in the human body such as cellular defense responses to infectious agents and a variety of cellular signaling pathways. While at low concentrations free radicals are involved in many significant metabolic reactions, high levels of free radicals can have deleterious effects on biomolecules like proteins, lipids, and DNA. Many physiological disorders such as diabetes, ageing, neurodegenerative diseases, and ischemia-reperfusion (I/R) injury are associated with oxidative stress.1 In particular, the deleterious effects caused by I/R injury developed during organ transplantation, cardiac infarct, and stroke have become the main cause of death in the United States and Europe.1,2 In this context, we synthesized and characterized a series of novel indole-amino acid conjugates as potential antioxidants for I/R injury. The synthesis of indole-phenol conjugate compounds is also discussed. Phenolic derivatives such as caffeic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), resveratrol, and its analogues are known for their significant antioxidative properties. A series of resveratrol analogues have been designed and synthesized as potential antioxidants. The radical scavenging mechanisms for potential antioxidants and assays for the in vitro evaluation of antioxidant activities are also discussed.

COMPARISON OF NON-HEATING PALEOINTENSITY TECHNIQUES USING BASALTS FROM LEMPTÉGY VOLCANO, FRANCE AND SYNTHETIC MAGNETITE-BEARING SAMPLES

Data of the strength of Earth’s magnetic field (paleointensity) in the geological past are crucial for understanding the geodynamo. Conventional paleointensity determination methods require heating a sample to a high temperature in one or more steps. Consequently, many rocks are unsuitable for these methods due to a heating-induced experimental alteration. Alternative non-heating paleointensity methods are investigated to assess their effectiveness and reliability using both natural samples from Lemptégy Volcano, France, and synthetic samples. Paleointensity was measured from the natural and synthetic samples using the Pseudo-Thellier, ARM, REM, REMc, REM’, and Preisach methods. For the natural samples, only the Pseudo-Thellier method was able to produce a reasonable paleointensity estimate consistent with previous paleointensity data. The synthetic samples yielded more successful estimates using all the methods, with the Pseudo-Thellier and ARM methods producing the most accurate results. The Pseudo-Thellier method appears to be the best alternative to the heating-based paleointensity methods.

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIO-OLIGOMERS

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications. To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The dissertation will present the details about the development of the techniques. Chapter 1 will make an introduction to oligodeoxynucleotides (ODNs), their synthesis and purification. Chapter 2 will describe the detailed studies of using the catching failure sequences by polymerization method to purify ODNs. Chapter 3 will describe the further optimization of the catching failure sequences by polymerization ODN purification technology to the level of practical use. Chapter 4 will present using the catching full-length sequence by polymerization method for ODN purification using acid-cleavable linker. Chapter 5 will make an introduction to peptides, their synthesis and purification. Chapter 6 will describe the studies using the catching full-length sequence by polymerization method for peptide purification.

SYNTHETIC OLIGODEOXYNUCLEOTIDE PURIFICATION VIA CATCHING BY POLYMERIZATION

Large quantities of pure synthetic oligodeoxynucleotides (ODNs) are important for preclinical research, drug development, and biological studies. These ODNs are synthesized on an automated synthesizer. It is inevitable that the crude ODN product contains failure sequences which are not easily removed because they have the same properties as the full length ODNs. Current ODN purification methods such as polyacrylamide gel electrophoresis (PAGE), reversed-phase high performance liquid chromatography (RP HPLC), anion exchange HPLC, and affinity purification can remove those impurities. However, they are not suitable for large scale purification due to the expensive aspects associated with instrumentation, solvent demand, and high labor costs. To solve these problems, two non-chromatographic ODN purification methods have been developed. In the first method, the full-length ODN was tagged with the phosphoramidite containing a methacrylamide group and a cleavable linker while the failure sequences were not. The full-length ODN was incorporated into a polymer through radical acrylamide polymerization whereas failure sequences and other impurities were removed by washing. Pure full-length ODN was obtained by cleaving it from the polymer. In the second method, the failure sequences were capped by a methacrylated phosphoramidite in each synthetic cycle. During purification, the failure sequences were separated from the full-length ODN by radical acrylamide polymerization. The full-length ODN was obtained via water extraction. For both methods, excellent purification yields were achieved and the purity of ODNs was very satisfactory. Thus, this new technology is expected to be beneficial for large scale ODN purification.

Sizing by Elutriation of Finely Ground Quartz and Pyrite

Elutriation, as a means of sorting mineral particles, has received marked attention during the last fifteen years. Its use in the ceramics industry for the sorting of clays was recognized even before this.

A Preliminary Study of the Separation of the Copper Sulfides from Sphalerite and the Effect of Certain Reagents on Some of the Pure Copper Minerals in Synthetic Mixtures

The problem of separating the copper sulfide minerals from sphalerite, in copper - zinc ores, has been a difficult one. This is largely due to the lack of adequate research and the small amount of data obtainable on the behavior of copper and zinc sulfide minerals in flotation circuits.

A feasibility study evaluating an in situ formed synthetic biodegradable membrane for guided bone regeneration in dogs

PURPOSE: The aim was (1) to evaluate the soft-tissue reaction of a synthetic polyethylene glycol (PEG) hydrogel used as a barrier membrane for guided bone regeneration (GBR) compared with a collagen membrane and (2) to test whether or not the application of this in situ formed membrane will result in a similar amount of bone regeneration as the use of a collagen membrane. MATERIAL AND METHODS: Tooth extraction and preparation of osseous defects were performed in the mandibles of 11 beagle dogs. After 3 months, 44 cylindrical implants were placed within healed dehiscence-type bone defects resulting in approximately 6 mm exposed implant surface. The following four treatment modalities were randomly allocated: PEG+autogenous bone chips, PEG+hydroxyapatite (HA)/tricalcium phosphate (TCP) granules, bioresorbable collagen membrane+autogenous bone chips and autogenous bone chips without a membrane. After 2 and 6 months, six and five dogs were sacrificed, respectively. A semi-quantitative evaluation of the local tolerance and a histomorphometric analysis were performed. For statistical analysis, repeated measures analysis of variance (ANOVA) and subsequent pairwise Student's t-test were applied (P<0.05). RESULTS: No local adverse effects in association with the PEG compared with the collagen membrane was observed clinically and histologically at any time-point. Healing was uneventful and all implants were histologically integrated. Four out of 22 PEG membrane sites revealed a soft-tissue dehiscence after 1-2 weeks that subsequently healed uneventful. Histomorphometric measurement of the vertical bone gain showed after 2 months values between 31% and 45% and after 6 months between 31% and 38%. Bone-to-implant contact (BIC) within the former defect area was similarly high in all groups ranging from 71% to 82% after 2 months and 49% to 91% after 6 months. However, with regard to all evaluated parameters, the PEG and the collagen membranes did not show any statistically significant difference compared with sites treated with autogenous bone without a membrane. CONCLUSION: The in situ forming synthetic membrane made of PEG was safely used in the present study, revealing no biologically significant abnormal soft-tissue reaction and demonstrated similar amounts of newly formed bone for defects treated with the PEG membrane compared with defects treated with a standard collagen membrane.

Bovine primary chondrocyte culture in synthetic matrix metalloproteinase-sensitive poly(ethylene glycol)-based hydrogels as a scaffold for cartilage repair

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

High-field 1H T1 and T2 NMR Relaxation Time Measurements of H2O in Homeopathic Preparations of Quartz, Sulfur and Copper Sulfate

Quantitative meta-analyses of randomized clinical trials investigating the specific therapeutic efficacy of homeopathic remedies yielded statistically significant differences compared to placebo. Since the remedies used contained mostly only very low concentrations of pharmacologically active compounds, these effects cannot be accounted for within the framework of current pharmacology. Theories to explain clinical effects of homeopathic remedies are partially based upon changes in diluent structure. To investigate the latter, we measured for the first time high-field (600/500 MHz) 1H T1 and T2 nuclear magnetic resonance relaxation times of H2O in homeopathic preparations with concurrent contamination control by inductively coupled plasma mass spectrometry (ICP-MS). Homeopathic preparations of quartz (10c–30c, n = 21, corresponding to iterative dilutions of 100−10–100−30), sulfur (13x–30x, n = 18, 10−13–10−30), and copper sulfate (11c–30c, n = 20, 100−11–100−30) were compared to n = 10 independent controls each (analogously agitated dilution medium) in randomized and blinded experiments. In none of the samples, the concentration of any element analyzed by ICP-MS exceeded 10 ppb. In the first measurement series (600 MHz), there was a significant increase in T1 for all samples as a function of time, and there were no significant differences between homeopathic potencies and controls. In the second measurement series (500 MHz) 1 year after preparation, we observed statistically significant increased T1 relaxation times for homeopathic sulfur preparations compared to controls. Fifteen out of 18 correlations between sample triplicates were higher for controls than for homeopathic preparations. No conclusive explanation for these phenomena can be given at present. Possible hypotheses involve differential leaching from the measurement vessel walls or a change in water molecule dynamics, i.e., in rotational correlation time and/or diffusion. Homeopathic preparations thus may exhibit specific physicochemical properties that need to be determined in detail in future investigations.

A New Synthetic Approach to 8-Aza Analogs of Prostaglandins

A new synthetic approach to (dl)-8-aza-13,14-dihydroprostanoic acid and its corresponding 11-hydroxy derivative is described